An acquired genetic mutation in somatic cells is sometimes highly responsible for, together with congenital genetic mutations in germ cells, susceptibility to a certain kind of disease, a therapeutic effect of a drug, strength of side effects and the like. In cancer cells, various genetic mutations occur at somatic cell level, and the mutations trigger cancer and greatly influence the response efficiency of molecular target drugs. In non-small cell lung cancer, for example, when EGFR gene is mutated, the response efficiency of gefitinib (trade name Iressa), which is one kind of molecular target drugs, becomes high and, when it is not mutated, the response efficiency becomes low. In colorectal cancer, when KRAS gene is mutated, the response efficiency of a molecular target drug cetuximab (trade name Erbitax) becomes low and, when it is not mutated, the response efficiency becomes high. Consequently, when a therapy using a molecular target drug is applied to these diseases, previous examination of the presence or absence of a mutation of the gene is increasingly required.
There are largely two kinds of methods for detecting a detection target nucleic acid (mutated gene) in somatic cells. One of them is a detection method including non-selectively amplifying a mutated gene and a detection non-target nucleic acid (wild-type gene), after which distinguishing the mutated gene from the wild-type gene. This detection method includes various methods such as a method utilizing electrophoresis, a method utilizing hybridization, and the like (see, for example, non-patent document 1). In most of the methods, however, detection of a slight amount of a mutated gene contained in a wild-type gene with sufficient sensitivity and accuracy is difficult. For example, a general detection method of mutated gene is the dideoxysequencing method. While the dideoxysequencing method permits detection of a mutated gene, present alone, with comparatively high sensitivity, when a mutated gene is contained in a trace amount in a wild-type gene, detection is possible only when the mutated gene is contained in about 10%. While the pyrosequencing method is superior to the dideoxysequencing method, the detection sensitivity thereof is reported to be about 5% (see, for example, non-patent document 2). Furthermore, a method including amplifying a mixed sample of mutated and wild-type genes by PCR, drawing a melting curve of a double stranded DNA of the amplification product thereof, and determining the ratio of the mutated gene from the difference in the melting curves of the mutated gene and the wild-type gene has been developed. Even by this method, the detection sensitivity of a mutated gene contained in a wild-type gene is about 5% (see, for example, non-patent document 3).
Another method for detecting a mutated gene in somatic cells is a method including distinguishing a wild-type gene from a mutated gene in the stage of gene amplification. Specifically, the method includes selective amplification of a mutated gene alone.
For example, it includes a method called “mutant-enriched PCR”, wherein a wild-type gene alone is cleaved with a restriction enzyme, and a non-cleaved mutated gene alone is amplified (e.g., non-patent document 4). This method is considered to be able to detect 1 molecule of a mutated gene in 106 molecules of a wild-type gene by repeating a reaction to amplify the mutated gene (see, for example, non-patent document 5). While this method is superior in high sensitivity as mentioned above, it cannot be applied to general diagnoses since the operation is highly complicated.
In an elongation reaction of primers in PCR and the like, a method including amplification by distinguishing a difference in a single base has been developed. This method is called “ARMS (amplification refractory mutation system)” (see, for example, non-patent document 6), “ASPCR (allele specific PCR)” (see, for example, non-patent document 7) and the like. This method is a superior method since it has comparatively high sensitivity, does not require an operation other than general PCR amplification reactions, can perform whole reactions in a closed system, is highly convenient, and is free of contamination. However, when a wild-type gene is amplified even once due to erroneous distinction of a single base, the risk of false-positivity becomes high since a wild-type gene is also amplified, like the amplification of mutated gene, in the subsequent amplification reaction. When this method is used, the reaction conditions, i.e., reaction temperature, salt concentration and the like, need to be controlled strictly, and the amount of templates needs to be precisely the same (see, for example, e.g., non-patent document 1), and therefore, the method is not suitable for clinical tests to examine an unspecified large number of samples, and diagnosis requiring high accuracy.
In the nucleic acid amplification techniques such as PCR and the like, another amplification method including distinguishing differences in the bases is a technique for inhibiting amplification of a wild-type gene by using an artificial oligonucleotide having a suitable kind and length of a structure completely complementary to a wild-type gene, or what is called a “clamp method”. A material for a clamp nucleic acid is required to, in a nucleic acid amplification process, (i) strongly hybridize to a wild-type gene, (ii) not strongly hybridize to a mutated gene, and (iii) resist decomposition in a nucleic acid amplification process. Accordingly, DNA and RNA composed of a natural material are not suitable in terms of hybridization capability and decomposition resistance, and artificial nucleic acids such as peptide nucleic acid (PNA) and locked nucleic acid (LNA) are exclusively used as a material for the clamp nucleic acid for the clamp technique (see, for example, e.g., non-patent documents 8-11 and patent documents 1, 2).
PNA is an artificial nucleic acid generally utilized as a fully-modified type and has properties desirable as a clamp nucleic acid, since it shows stronger hybridization capability to a DNA strand having a complementary structure than does natural DNA, shows good difference from the hybridization capability to a DNA strand having a one-base mismatch sequence, and free of degradation by nucleases. As such, it is frequently used as a clamp nucleic acid for the analysis and detection of various mutations of various genes. On the other hand, LNA is an artificial nucleic acid utilized as a partially modified type. Since every modification of one unit strikingly enhances the hybridization capability, it can be controlled by the length and modification mode to acquire suitable hybridization capability. As such, it is possible to afford a clamp effect by using a comparatively short LNA oligonucleotide.
However, the above-mentioned clamp nucleic acids each have difficulties. Even if PNA “shows higher hybridization capability than natural DNA”, the level thereof is limited and the clamp nucleic acid needs to be elongated to acquire good hybridization capability and exhibit a sufficient clamp effect. In addition, while LNA is completely free of problems relating to the hybridization capability, its chemical stability in the amplification process poses problems since resistance thereof to the nucleases is not very strong and, comprehensively, LNA is not entirely superior in clamp capacity.
The present inventors have developed, after the development of 2′,4′-BNA (LNA), which is a crosslinking structure type novel artificial nucleic acid (first generation BNA) (e.g., patent document 3), BNAs of the second generation and thereafter such as 3′-amino-2′,4′-BNA, 5′-amino-2′,4′-BNA, 2′,4′-BNAccc, 2′,4′-BNANC and the like (e.g., patent documents 4-7).